Alpha-amylase variant with altered properties

Abstract

The present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits altered properties relative to the parent alpha-amylase.

1 - 21 . (canceled) 
     
     
         22 . A method for producing a variant bacterial a-amylase polypeptide, comprising:
 introducing into the amino sequence of a parent bacterial a-amylase polypeptide a mutation at an amino acid residue which alters substrate binding;   wherein the mutation alters the binding of substrate to the variant bacterial a-amylase polypeptide compared to the parental bacterial a-amylase polypeptide, wherein the variant retains its alpha-amylase activity.   
     
     
         23 . The method of  claim 22 , wherein the amino acid residues which alter substrate binding corresponds to amino acid residues 1-6, 36, 38, 91-93, 95-97, 225-226, 249-255, 257, 278-282, 309-320, 354, 355-357, 358, 391, and 395-402, referring to SEQ ID NO: 8 for numbering, or amino acid residues 1-6, 37, 39, 92-93, 95-98, 227-229, 252-255, 257-260, 281-285, 312-323, 357-362, 391, and 395-402, referring to SEQ ID NO: 6 for numbering. 
     
     
         24 . The method of  claim 22 , wherein the mutation is in an amino acid residue corresponding to amino acid residues 92, 249, 252, 254, 315, 316, 318, or 319, referring to SEQ ID NO: 8 for numbering, or to amino acid residues 93, 252, 255, 257, 318, 319 or 322, referring to SEQ ID NO: 6 for numbering. 
     
     
         25 . The method of  claim 22 , wherein the mutation is the substitution of the wild-type residue at a position corresponding to R249 or K254 with a different amino acid residue, referring to SEQ ID NO: 8 for numbering, or at a position corresponding to R252 or K257, referring to SEQ ID NO: 6 for numbering. 
     
     
         26 . The method of  claim 22 , wherein the mutation is the substitution of the wild-type residue at a position corresponding to position 92 in SEQ ID NO: 8, or to position 93 in SEQ ID NO: 6, with L, the substitution of the wild-type residue at a position corresponding to position 249 in SEQ ID NO: 8, or to position 252 in SEQ ID NO: 6, with A, C, D, E, F, G, H, L, M, N, Q, S, T, or W, the substitution of the wild-type residue at a position corresponding to position 252 in SEQ ID NO: 8, or to position 255 in SEQ ID NO: 6, with H, K, W, or Y, the substitution of the wild-type residue at a position corresponding to position 254 in SEQ ID NO: 8, or to position 257 in SEQ ID NO: 6, with A, E, T, or V, the substitution of the wild-type residue at a position corresponding to position 315 in SEQ ID NO: 8, or to position 318 in SEQ ID NO: 6, with C or R, the substitution of the wild-type residue at a position corresponding to position 316 in SEQ ID NO: 8, or to position 319 in SEQ ID NO: 6, with A, F, H, K, Q, or R, the substitution of the wild-type residue at a position corresponding to position 318 in SEQ ID NO: 8, or to position 321 in SEQ ID NO: 6, with A, H, M, N, or P, or the substitution of the wild-type residue at a position corresponding to position 319 in SEQ ID NO: 8, or to position 322 in SEQ ID NO: 6, with D, G, I, or T. 
     
     
         27 . The method of  claim 22 , wherein the mutation corresponds to S92L, R249A, R249C, R249D, R249E, R249F, R249G, R249L, R249M, R249N, R249Q, R249S, R249T, R249W, T252H, T252K, T252W, or T252Y, K254A, K254E, K254T, or K254V, K315C or K315R, H316A, H316F, H316K, H316Q, or H316R, and/or L318A, L318H, L318M, L318N, or L318P, referring to SEQ ID NO: 8 for numbering, or corresponds to A93L, R252A, R252C, R252D, R252E, R252F, R252G, R252L, R252M, R252N, R252Q, R252S, R252T, R252W, T255H, T255K, T255W, T255Y, K257A, K257E, K257T, or K257V, D318C, D318R, Q319A, Q319F, Q319H, Q319K, or Q319R and/or T321A, T321H, T321M, T321N, or T321P, referring to SEQ ID NO: 6 for numbering. 
     
     
         28 . The method of  claim 22 , wherein the variant further comprises wild-type amino acid residues at one or more positions corresponding to K88, E250, K251, K306, S314, E355, A398, H400, and D401, referring to SEQ ID NO: 8 for numbering, or at one or more positions corresponding to Q89, S253, Q254, T309, K317, Q358, A398, H400 and D401, referring to SEQ ID NO: 6 for numbering. 
     
     
         29 . The method of  claim 22 , wherein the variant further comprises wild-type amino acid residues at one or more positions corresponding to N4, G5, T38, A93, D94, 195, N96, V97, F228, G253, V313, P317, S320, L352, T353, R354, G357, and Y394, A395, Y396, G397, and Q399 in SEQ ID NO: 8, referring to SEQ ID NO: 8 for numbering, or at one or more positions corresponding to N5, G6, T39, A94, G95, Q97, V98, F231, G256, M316, P320, A323, L355, R357, G360, Y394, A395, Y396, G397, and Q399 in SEQ ID NO: 6, referring to SEQ ID NO: 6 for numbering. 
     
     
         30 . The method of  claim 22 , wherein the variant further comprises N at position 88, A at position 250, A at position 314, Sat position 355, or R at position 400, or D at position 401, referring to SEQ ID NO: 8 for numbering, or further comprises N at position 89, A at position 253, A at position 317, Sat position 358, Rat position 400, or D at position 401, referring to SEQ ID NO: 6 for numbering. 
     
     
         31 . The method of  claim 22 , wherein the variant further comprises N at position 4, G at position 5, T at position 38, R at position 93, D at position 94, I at position 95, N at position 96, V at position 97, Y at position 228, G at position 253, V at position 313, P at position 317, L at position 352, T at position 353, R at position 354, G at position 357, and Y at position 394, A at position 395, Y at position 396, G at position 397, and Q at position 399, referring to SEQ ID NO: 8 for numbering, or further comprises N at position 5, G at position 6, T at position 39, A at position 94, G at position 95, M at position 96, Q at position 97, V at position 98, Y at position 231, G at position 254, M at position 313, P at position 318, L at position 355, Tat position 356, R at position 357, G at position 360, and Y at position 394, A at position 395, Y at position 396, G at position 397, and Q at position 399, referring to SEQ ID NO: 6 for numbering. 
     
     
         32 . The method of  claim 22 , wherein the variant exhibits improved starch liquefaction, starch saccharification, or cleaning performance compared to the parent. 
     
     
         33 . The method of  claim 22 , wherein the variant exhibits increased hydrolysis activity on an amylose substrate compared to the parent.